ABSTRACT: Ovarian cancer is a deadly disease with the 3rd-highest mortality:incidence ratio of all cancers. High-grade serous cancer (HGSC) is the most aggressive ovarian cancer subtype for which we have seen only little or no improvement in patient survival. Thus there is a clear unmet need to identify and develop new therapeutic targets in HGSC. We recently showed that the stem cell regulatory factor EGFL6 is a critical regulator of ALDH+ HGSC cancer stem-like cells (CSC), cells associated with therapeutic resistance. EGFL6 promotes the migration and asymmetric division of ALDH+HGSC CSC, while EGFL6 knockdown in HGSC cancer cells leads to loss of stemness and dramatically reduced tumor growth in mice. We therefore hypothesize that EGFL6 is a promising therapeutic target for HGSC. Understanding the therapeutic potential EGFL6-directed agents will require greater understanding of EGFL6's roles in ovarian cancer cell biology. The EGFL6 receptor on cancer cells is unknown. As disruption of ligand/receptor signaling has proven a very effective therapeutic mechanism in other pathways, we propose SA1: To Identify the EGFL6 receptor and characterize the EGFL6 signaling complex. In addition to regulating HGSC CSC, EGFL6 is an important regulator of normal stem cells. Mutated normal stem cells are a proposed source of cancer initiating cells. We hypothesize EGFL6 as a regulator of both normal stem cells and HGSC CSC may be essential for ovarian cancer initiation and growth. We therefore propose SA2: To evaluate the role of EGFL6 in cancer initiation. We will use a novel genetic mouse model of HGSC to assess (i) the impact of EGFL6 knockout or (ii) the effect of EGFL6 neutralizing antibodies on HGSC initiation and growth. We have shown that the murine EGFL6 neutralizing antibody we developed has excellent therapeutic activity versus human cancer cell lines in mice. To translate these studies into clinical trials we developed a a panel of humanized EGFL6-blocking antibodies (hEGFL6-Ab). We propose SA3: To validate hEGFL6-Ab and determine if EGFL6 expression by patients' tumors predicts response to anti-EGFL6 therapy. Using in vitro assays and a novel humanized stroma- patient derived xenograft model we will identify the most effective hEGFL6-Ab. We hypothesize that patients whose tumor cells express EGFL6 and/or exhibit EGFL6 pathway activation will be most responsive to such therapy. Using expression analysis of hEGFL6-Ab responsive and non-responsive tumors, we propose to generate an algorithm to predict patients' responses to anti-EGFL6 therapy. IMPACT: These studies will 1) define the EGFL6 signaling cascade in ovarian cancer cells, 2) define requirements for cancer cell EGFL6 expression in HGSC initiation and growth, and 3) create and validate a novel humanized anti-EGFL6-Ab and an accompanying algorithm that identifies/stratifies patients who are most apt to respond to such therapy. Ultimately, the project will produce a promising therapeutic agent positioned for first-in-human clinical trials in ovarian cancer.